Abstract 1404 Developing an anti-CD8 - BiTE to eliminate factor VIII-specific B Cells

Factor VIII (FVIII) is an essential component of blood coagulation. It is encoded by the F8 gene of the human genome, and loss of function mutations result in hemophilia type A (HA), in which blood cannot clot properly. There is no cure for HA, but patients can receive regular infusions of FVIII to replace the deficiency. Replacement therapy allows most patients to lead a normal life, but 20-30% of patients with a FVIII deficiency will develop an immune response against replacement therapy, producing antibodies that neutralize the effect. We have developed a bispecific T-cell engager (BiTE) designed to eliminate FVIII-specific B cells that produce neutralizing antibodies. The BiTE links the highly immunogenic C2 domain from FVIII to bind anti-C2 lymphocytes and an anti-CD8 single-chain variable fragment (scFv) to bind cytotoxic T-cells. I constructed the recombinant gene for this C2/anti-CD8 fusion protein utilizing Gibson assembly, then expressed the protein in E. coli, refolded it, and purified it by affinity chromatography. A B cell ELISpot assay in the presence of PBMCs will be utilized to examine the ability of the BiTE to recruit CD8-positive T cells to eliminate anti-C2 hybridomas. This project could potentially tolerize refractory HA patients to transfusions of FVIII, improving blood coagulation and overall quality of life. I would like to express my profound gratitude to Towson University Fisher College of Science and Mathematics for the undergraduate research grant, the National Institutes of Health for the Exploratory/Developmental Research Grant Award (R21 AI152318), and collaborators at Uniformed Services University.